Adaptation and Cryptic Pseudogenization in Penguin Toll-Like Receptors.

Penguins (Sphenisciformes) are an iconic order of flightless, diving seabirds distributed across a large latitudinal range in the Southern Hemisphere. The extensive area over which penguins are endemic is likely to have fostered variation in pathogen pressure, which in turn will have imposed differential selective pressures on the penguin immune system. At the front line of pathogen detection and response, the Toll-like receptors (TLRs) provide insight into host evolution in the face of microbial challenge. TLRs respond to conserved pathogen-associated molecular patterns and are frequently found to be under positive selection, despite retaining specificity for defined agonist classes. We undertook a comparative immunogenetics analysis of TLRs for all penguin species and found evidence of adaptive evolution that was largely restricted to the cell surface-expressed TLRs, with evidence of positive selection at, or near, key agonist-binding sites in TLR1B, TLR4, and TLR5. Intriguingly, TLR15, which is activated by fungal products, appeared to have been pseudogenized multiple times in the Eudyptes spp., but a full-length form was present as a rare haplotype at the population level. However, in vitro analysis revealed that even the full-length form of Eudyptes TLR15 was nonfunctional, indicating an ancestral cryptic pseudogenization prior to its eventual disruption multiple times in the Eudyptes lineage. This unusual pseudogenization event could provide an insight into immune adaptation to fungal pathogens such as Aspergillus, which is responsible for significant mortality in wild and captive bird populations.

Cannabinoid receptor 2 evolutionary gene loss makes parrots more susceptible to neuroinflammation.

In vertebrates, cannabinoids modulate neuroimmune interactions through two cannabinoid receptors (CNRs) conservatively expressed in the brain (CNR1, syn. CB1) and in the periphery (CNR2, syn. CB2). Our comparative genomic analysis indicates several evolutionary losses in the CNR2 gene that is involved in immune regulation. Notably, we show that the CNR2 gene pseudogenized in all parrots (Psittaciformes). This CNR2 gene loss occurred because of chromosomal rearrangements. Our positive selection analysis suggests the absence of any specific molecular adaptations in parrot CNR1 that would compensate for the CNR2 loss in the modulation of the neuroimmune interactions. Using transcriptomic data from the brains of birds with experimentally induced sterile inflammation we highlight possible functional effects of such a CNR2 gene loss. We compare the expression patterns of CNR and neuroinflammatory markers in CNR2-deficient parrots (represented by the budgerigar, Melopsittacus undulatus and five other parrot species) with CNR2-intact passerines (represented by the zebra finch, Taeniopygia guttata). Unlike in passerines, stimulation with lipopolysaccharide resulted in neuroinflammation in the parrots linked with a significant upregulation of expression in proinflammatory cytokines (including interleukin 1 beta (IL1B) and 6 (IL6)) in the brain. Our results indicate the functional importance of the CNR2 gene loss for increased sensitivity to brain inflammation.

Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells.

Gene targeting by homologous recombination or by sequence-specific nucleases allows the precise modification of genomes and genes to elucidate their functions. Although gene targeting has been used extensively to modify the genomes of mammals, fish, and amphibians, a targeting technology has not been available for the avian genome. Many of the principles of humoral immunity were discovered in chickens, yet the lack of gene targeting technologies in birds has limited biomedical research using this species. Here we describe targeting the joining (J) gene segment of the chicken Ig heavy chain gene by homologous recombination in primordial germ cells to establish fully transgenic chickens carrying the knockout. In homozygous knockouts, Ig heavy chain production is eliminated, and no antibody response is elicited on immunization. Migration of B-lineage precursors into the bursa of Fabricius is unaffected, whereas development into mature B cells and migration from the bursa are blocked in the mutants. Other cell types in the immune system appear normal. Chickens lacking the peripheral B-cell population will provide a unique experimental model to study avian immune responses to infectious disease. More generally, gene targeting in avian primordial germ cells will foster advances in diverse fields of biomedical research such as virology, stem cells, and developmental biology, and provide unique approaches in biotechnology, particularly in the field of antibody discovery.

Development and characterization of a CRISPR/Cas9-mediated RAG1 knockout chicken model lacking mature B and T cells.

Although birds have been used historically as a model animal for immunological research, resulting in remarkable achievements, immune cell development in birds themselves has yet to be fully elucidated. In this study, we firstly generated an immunodeficient chicken model using a CRISPR/Cas9-mediated recombination activating gene 1 (RAG1) knockout, to investigate avian-specific immune cell development. Unlike previously reported immunoglobulin (Ig) heavy chain knockout chickens, the proportion and development of B cells in both RAG1 +/- and RAG1 -/- embryos were significantly impaired during B cell proliferation (embryonic day 16 to 18). Our findings indicate that, this is likely due to disordered B cell receptor (BCR)-mediated signaling and interaction of CXC motif chemokine receptor (CXCR4) with CXCL12, resulting from disrupted Ig V(D)J recombination at the embryonic stage. Histological analysis after hatching showed that, unlike wild-type (WT) and RAG1 +/- chickens, lymphatic organs in 3-week old RAG1 -/- chickens were severely damaged. Furthermore, relative to WT chickens, RAG1+/- and RAG1-/- birds had reduced serum Igs, fewer mature CD4+ and CD8+ T lymphocytes. Furthermore, BCR-mediated B cell activation in RAG1 +/- chickens was insufficient, leading to decreased expression of the activation-induced deaminase (AID) gene, which is important for Ig gene conversion. Overall, this immunodeficient chicken model underlines the pivotal role of RAG1 in immature B cell development, Ig gene conversion during embryonic stages, and demonstrates the dose-dependent regulatory role of RAG1 during immune cell development. This model will provide ongoing insights for understanding chicken immune system development and applied in the fields of immunology and biomedical science.

Early Life Inoculation With Adult-Derived Microbiota Accelerates Maturation of Intestinal Microbiota and Enhances NK Cell Activation in Broiler Chickens.

Studies in mammals, including chickens, have shown that the development of the immune system is affected by interactions with intestinal microbiota. Early life microbial colonization may affect the development of innate and adaptive immunity and may contribute to lasting effects on health and resilience of broiler chickens. We inoculated broiler chickens with adult-derived-microbiota (AM) to investigate their effects on intestinal microbiota composition and natural killer (NK) cells, amongst other immune cells. We hypothesized that AM inoculation directly upon hatch (day 0) would induce an alteration in microbiota composition shortly after hatch, and subsequently affect (subsets of) intestinal NK cells and their activation. Microbiota composition of caecal and ileal content of chickens of 1, 3, 7, 14, 21, and 35 days of age was assessed by sequencing of 16S ribosomal RNA gene amplicons. In parallel, subsets and activation of intestinal NK cells were analyzed by flow cytometry. In caecal content of 1- and 3-day-old AM chickens, a higher alpha-diversity (Faith's phylogenetic diversity) was observed compared to control chickens, whereas ileal microbiota were unaffected. Regarding beta-diversity, caecal microbiota profiles could be clustered into three distinct community types. Cluster A represented caecal microbiota of 1-day-old AM chickens and 1- and 3-day-old control chickens. Cluster B included microbiota of seven of eight 3- and 7-day-old AM and 7-day-old control chickens, and cluster C comprised microbiota of all chickens of 14-days and older, independent of inoculation. In 3-day-old AM chickens an increase in the percentages of intestinal IL-2Rα+NK cells and activated NK cells was observed compared to control chickens of the same age. In addition, an increase in relative numbers of intestinal cytotoxic CD8αα+T cells was observed in 14- and 21-day-old AM chickens. Taken together, these results indicate that early exposure to AM shapes and accelerates the maturation of caecal microbiota, which is paralleled by an increase in IL-2Rα+NK cells and enhanced NK cell activation. The observed association between early life development of intestinal microbiota and immune system indicates possibilities to apply microbiota-targeted strategies that can accelerate maturation of intestinal microbiota and strengthen the immune system, thereby improving the health and resilience of broiler chickens.

From Chickens to Humans: The Importance of Peptide Repertoires for MHC Class I Alleles.

In humans, killer immunoglobulin-like receptors (KIRs), expressed on natural killer (NK) and thymus-derived (T) cells, and their ligands, primarily the classical class I molecules of the major histocompatibility complex (MHC) expressed on nearly all cells, are both polymorphic. The variation of this receptor-ligand interaction, based on which alleles have been inherited, is known to play crucial roles in resistance to infectious disease, autoimmunity, and reproduction in humans. However, not all the variation in response is inherited, since KIR binding can be affected by a portion of the peptide bound to the class I molecules, with the particular peptide presented affecting the NK response. The extent to which the large multigene family of chicken immunoglobulin-like receptors (ChIRs) is involved in functions similar to KIRs is suspected but not proven. However, much is understood about the two MHC-I molecules encoded in the chicken MHC. The BF2 molecule is expressed at a high level and is thought to be the predominant ligand of cytotoxic T lymphocytes (CTLs), while the BF1 molecule is expressed at a much lower level if at all and is thought to be primarily a ligand for NK cells. Recently, a hierarchy of BF2 alleles with a suite of correlated properties has been defined, from those expressed at a high level on the cell surface but with a narrow range of bound peptides to those expressed at a lower level on the cell surface but with a very wide repertoire of bound peptides. Interestingly, there is a similar hierarchy for human class I alleles, although the hierarchy is not as wide. It is a question whether KIRs and ChIRs recognize class I molecules with bound peptide in a similar way, and whether fastidious to promiscuous hierarchy of class I molecules affect both T and NK cell function. Such effects might be different from those predicted by the similarities of peptide-binding based on peptide motifs, as enshrined in the idea of supertypes. Since the size of peptide repertoire can be very different for alleles with similar peptide motifs from the same supertype, the relative importance of these two properties may be testable.

Involvement of T Cell Immunity in Avian Coccidiosis.

Avian coccidiosis is caused by Eimeria, which is an intracellular apicomplexan parasite that invades through the intestinal tract to cause devastating disease. Upon invasion through the intestinal epithelial cells, a strong inflammatory response is induced that results in complete villous destruction, diarrhea, hemorrhage, and in severe cases, death. Since the life cycle of Eimeria parasites is complex and comprises several intra- and extracellular developmental stages, the host immune responses are diverse and complex. Interferon-γ-mediated T helper (Th)1 response was originally considered to be the predominant immune response in avian coccidiosis. However, recent studies on other avian T cell lineages such as Th17 and T regulatory cells have implicated their significant involvement in maintaining gut homeostasis in normal and disease states including coccidiosis. Therefore, there is a need to understand better their role in coccidiosis. This review focuses on research findings concerning the host immune response induced by avian coccidiosis in the context of T cell immunity, including expression of T-cell-related cytokines and surface molecules that determine the phenotype of T lymphocytes.

Identification and structural composition of the blood-spleen barrier in chickens.

To identify the existence and composition of the blood-spleen barrier (BSB) in chickens, the microanatomical features of the spleen were investigated by light and transmission electron microscopy, intravenous injection of ink, acid phosphatase reaction, and silver impregnation. The results showed that the white pulp in chicken spleen consists of lymphoid nodules, periarteriolar lymphatic sheaths (PALS) and periellipsoidal lymphatic sheaths (PELS). There was no evidence for the presence of a marginal zone. The splenic ellipsoid was a unique structure, which functioned as a barrier for filtering and phagocytosis. Uptake of carbon particles was limited to the ellipsoid and PELS, 60 min after injection of carbon particles. Reticular fibres were densely distributed in the ellipsoid and extended into the PELS. Ellipsoid-associated cells (EACs), reticular cells and macrophages were acid phosphatase positive. The sheathed capillaries, surrounded by the ellipsoid, were similar to high endothelial venules (HEVs). These findings suggest that the BSB of chickens is present in the ellipsoid and PELS, protecting the spleen from invasion from circulating pathogens. The BSB was a reticular framework, between the arterial and venous vessels, which included cuboidal-shaped endothelial cells, supporting cells, EACs, macrophages, reticular cells and fibres. Lymphocyte migration into the spleen parenchyma is most likely via the HEV-like vessels. These research findings contribute to better understanding of avian immunology and provide an insight into evolutionary differences in the immune system.

Assessing the efficacy of an inactivated chicken anemia virus vaccine.

BACKGROUND: Chicken anemia virus (CAV) is an immunosuppressive virus that causes chicken infectious anemia (CIA) which is a highly contagious avian disease. CAV causes major economic losses in the poultry industry worldwide. The current CAV vaccine is a live attenuated strain administered in the drinking water that risks horizontal infection of other chickens. The purpose of this study was to develop a novel vaccine against CAV that can be administered safely using a highly pathogenic isolate inactivated with ß-propiolactone hydrolysis that would protect chicks from CAV. METHODS: Hens were vaccinated twice intramuscularly with a novel CAV GD-G-12 inactivated vaccine and the humoral immune responses of the hens and offspring were monitored by ELISA. A heterologous intramuscular challenge using the CAV strain GD-E-12 was conducted in the chicks hatched from vaccinated or unvaccinated hens. RESULTS: The vaccine strain, GD-G-12, was shown to be highly pathogenic prior to inactivation evidenced by thymic atrophy and bleeding, and weight loss. The inactivated vaccine was considered safe and showed no signs of pathogenicity. High titers of CAV specific antibodies were detected in the vaccinated hens and in their chicks, indicating vertical transfer of maternal antibodies. Furthermore, the chicks hatched from vaccinated hens were resistant to a heterologous CAV challenge and showed no signs of weight loss and thymic atrophy and bleeding. CONCLUSION: Our studies are proof of principle that inactivated GD-G-12 might be a novel vaccine candidate to prevent CAV infection, and highlight the utility of using an inactivated virus for this vaccine.

Duck TRIM27-L enhances MAVS signaling and is absent in chickens and turkeys.

Wild waterfowl, including mallard ducks, are the natural reservoir of avian influenza A virus and they are resistant to strains that would cause fatal infection in chickens. Here we investigate potential involvement of TRIM proteins in the differential response of ducks and chickens to influenza. We examine a cluster of TRIM genes located on a single scaffold in the duck genome, which is a conserved synteny group with a TRIM cluster located in the extended MHC region in chickens and turkeys. We note a TRIM27-like gene is present in ducks, and absent in chickens and turkeys. Orthologous genes are predicted in many birds and reptiles, suggesting the gene has been lost in chickens and turkeys. Using quantitative real-time PCR (qPCR) we show that TRIM27-L, and the related TRIM27.1, are upregulated 5- and 9-fold at 1 day post-infection with highly pathogenic A/Vietnam/1203/2004. To assess whether TRIM27.1 or TRIM27-L are involved in modulation of antiviral gene expression, we overexpressed them in DF1 chicken cells, and neither show any direct effect on innate immune gene expression. However, when co-transfected with duck RIG-I-N (d2CARD) to constitutively activate the MAVS pathway, TRIM27.1 weakly decreases, while TRIM27-L strongly activates innate immune signaling leading to increased transcription of antiviral genes MX1 and IFN-ß. Furthermore, when both are co-expressed, the activation of the MAVS signaling pathway by TRIM27-L over-rides the inhibition by TRIM27.1. Thus, ducks have an activating TRIM27-L to augment MAVS signaling following RIG-I detection, while chickens lack both TRIM27-L and RIG-I itself.

Alteration in lymphocytes responses, cytokine and chemokine profiles in laying hens infected with Salmonella Typhimurium.

Salmonella Typhimurium has been reported to contaminate egg production across the world, but the exact nature of the immune mechanisms protective against Salmonella infection in laying hens has not been characterized at the molecular level. The experiment was conducted to determine Salmonella colonization and lymphocytes subpopulation in the ileum and spleen, and the mRNA expression of pro-inflammatory cytokines [interleukin (IL)-1ß and IL-6], chemokine IL-8, and T helper (Th)1/Th2 cytokines [Interferon (IFN)-γ, IL-12 and IL-18; IL-4 and IL-10 respectively] in the cecal tonsil and spleen of Salmonella challenged hens. Forty Salmonella-free laying hens were challenged orally with Salmonella Typhimurium or phosphate-buffered saline (PBS; control). The Salmonella challenged or non-challenged hens (n=10) were sacrificed at 2 and 7 days post-infection (DPI). The lymphocyte subpopulation was determined via flow cytometric analysis in the ileum and spleen. The cecal tonsil and spleen samples were collected for mRNA expression through quantitative-RT-PCR. The Salmonella counts were higher (P<0.05) in the ileum than that in the spleen at 2 and 7DPI, and were higher (P<0.05) at 7DPI than that at 2DPI in the spleen. Salmonella challenge increased (P<0.05) ileal CD4+ and CD8α+ cells ratios at 2 and 7DPI, whereas it increased (P<0.05) splenic CD8α+ cells ratio only at 7DPI. The magnitude of increase in ileal CD8α+ cells ratio was higher (P<0.05) than that in CD4+ cells ratio. The mRNA expression of IL-1ß, IL-6, IL-8, IFN-γ, IL-12 and IL-18 were significantly up-regulated in the cecal tonsil of Salmonella challenged hens, and the magnitude of increases in IL-6, IL-8 and IL-12 were significantly higher at 7DPI than that at 2DPI. However, Salmonella challenge increased (P<0.05) the mRNA expression of IL-1ß, IL-10 and IL-18 at 2 and 7DPI, and IL-8 and IFN-γ mRNA only at 7DPI in the spleen. These findings demonstrated that there appeared the induction of cellular immune responses, and a Th1-cytokines reaction in the intestine and spleen of laying hens infected with Salmonella Typhimurium.